identification and quantification of surface wax compounds covering aerial organs of selected plant

251 Pages·2016·5.68 MB·English

IDENTIFICATION AND QUANTIFICATION OF SURFACE WAX COMPOUNDS
COVERING AERIAL ORGANS OF SELECTED PLANT SPECIES
by

Radu C. Racovita

Dipl.Eng., Politehnica University, 2008
M.Sc., The University of Western Ontario, 2011

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY
in
THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES
(Chemistry)

THE UNIVERSITY OF BRITISH COLUMBIA
(Vancouver)

August 2016

© Radu C. Racovita, 2016
Abstract

The cuticle is an external barrier of aerial plant organs that prevents dessication. It is composed
of hydrophobic waxes, i.e. complex mixtures of very-long-chain aliphatics, alicyclics and
aromatics, lying on top of (epicuticular) and in between (intracuticular) a polyester matrix known
as cutin. Wax compositions vary greatly between plant species, organs and tissues, both
qualitatively and quantitatively. This thesis describes the identification and quantification of
cuticular waxes of three plant species, including structure elucidation of novel compounds, chain
length profiling, and wax compound distributions between intracuticular and epicuticular
compartments for the first two species.
Leaves of Aloe arborescens were found covered with 15 μg/cm2 wax on the adaxial side and 36
μg/cm2 on the abaxial side, with 3:2 and 1:1 ratios between epicuticular and intracuticular wax
layers on each side, respectively. Along with ubiquitous wax compounds, three homologous
series were identified as novel 3-hydroxy fatty acids (predominantly C ), their methyl esters
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(predominantly C ), and 2-alkanols (predominantly C ), and their biosynthetic pathways were
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hypothesized based on structural similarities and homolog distributions.
The adaxial side of young and old Phyllostachys aurea leaves was found covered with 1.7 to 1.9
μg/cm2 each of epi- and intracuticular waxes. In addition to typical aliphatics and alicyclics,
novel primary amides were identified, with their chain length profile peaking at C , and found
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exclusively in the epicuticular waxes, hence near the true plant surface.
Flag leaves and peduncles of Triticum aestivum cv. Bethlehem were found covered with 16 and
49 μg/cm2 wax, respectively, dominated by 1-alkanols in the case of the former and β-diketones
and hydroxy-β-diketones for the latter. Along with previously reported wax classes, numerous
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new classes were identified as homologous series: 2-alkanol esters, benzyl esters, phenethyl
esters, p-hydroxyphenethyl esters, secondary alcohols, primary/secondary diols and their esters,
hydroxy- and oxo-2-alkanol esters, 4-alkylbutan-4-olides, internally methyl-branched alkanes,
and 2,4-ketols. Other new compounds were found as single homologs: C 2,4-diketone, C mid-
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chain β-ketols, C mid-chain α-ketols and α-diketone, as well as C mid-chain ketones.
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Biosynthetic pathways are proposed in the thesis for the new compounds, based on common
structural features and matching chain length patterns between related compound classes.

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Preface

A version of Chapter 2 has been published: Racovita, R.C., Peng, C., Awakawa, T., Abe, I.,
Jetter, R. Phytochemistry 2015, 113, 183-194. Reproduced with permission from Elsevier. I
conducted all experiments, interpreted results and wrote most of the manuscript; co-authors
helped with species selection (T.A.,I.A.), synthesized prior standards (C.P.), and contributed to
data interpretation and writing (R.J.).

A version of Chapter 3 has been published: Racovita, R.C., Jetter, R. Phytochemistry 2016,
Online publication http://dx.doi.org/10.1016/j.phytochem.2016.06.005. Reproduced with
permission from Elsevier. I performed all experiments and data interpretation and wrote most of
the manuscript; the co-author (R.J.) performed statistical analysis and contributed to data
interpretation and writing.

A version of Chapter 4 has been published: Racovita, R.C., Hen-Avivi, S., Fernandez-Moreno,
J.-P., Granell, A., Aharoni, A., Jetter, R. Phytochemistry 2016, Online publication
http://dx.doi.org/10.1016/j.phytochem.2016.05.003. Reproduced with permission from Elsevier.
I carried out most experiments and data interpretation, as well as wrote most of the manuscript;
co-authors grew plants and supplied surface wax extracts (S.H.A., J.P.F.M., A.G., A.A.), and
contributed to data interpretation and writing (R.J.).

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A version of Chapter 5 has been submitted for publication. I performed the experiments,
interpreted results and wrote most of the manuscript; the co-author, Dr. Reinhard Jetter,
contributed to figure editing, data interpretation and writing.

A version of Chapter 6 has been submitted for publication. I performed the experiments,
interpreted results and wrote most of the manuscript; the co-author, Dr. Reinhard Jetter,
contributed to figure editing, data interpretation and writing.

Certain paragraphs from Chapter 7 are concluding remarks from the manuscripts mentioned
above. These paragraphs were written by me and edited by Dr. Reinhard Jetter.

The experiments, data analysis, interpretation and writing in the remaining chapters were my
own contribution in close consultation with my supervisor, Dr. Reinhard Jetter.
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Table of contents

Abstract...………………………………………………………………………………………...ii
Preface…………………………………………………………………………………………....iv
Table of contents……………………………………………………………………...………....vi
List of tables……………………………………………………………………………………. xii
List of figures………………………………………………………………………………….. xiii
List of abbreviations ................................................................................................................. xvii
Acknowledgements .................................................................................................................... xix
Dedication .....................................................................................................................................xx
Chapter 1: Introduction to the plant cuticle................................................................................1
1.1. The plant cuticle: An adaptation to a dry environment…………………………..…..1
1.2. Chemical diversity of cuticle constituents…………………………………………..….1
1.2.1. Cutin/Cutan…………..……………………………………………………………1
1.2.2. Cuticular waxes……………………………………………………………………3
1.2.2.1. Aliphatics…………………..………………………………………………...3
1.2.2.2. Alicyclics……………………………..………………………………………5
1.2.2.3. Aromatics……………………..……………………………………………...7
1.3. Stratification of cuticle constituents………………………..…………………………..8
1.4. Functions of the plant cuticle…………………………..……………………………...10
1.5. Biosynthesis of VLC aliphatic cuticular waxes…………..…………………………..13
1.5.1. Biosynthesis of ubiquitous aliphatic waxes……………………………………..13
1.5.2. Biosynthesis of β-diketones in Poaceae…………………………………………16

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1.6. Objectives………………..……………………………………………………………...17
Chapter 2: Very-long-chain 3-hydroxy fatty acids, 3-hydroxy fatty acid methyl esters and
2-alkanols from cuticular waxes of Aloe arborescens leaves ....................................................20
2.1. Introduction………………..…………………………………………………………...20
2.2. Experimental……………..…………………………………………………………….23
2.2.1. Preparation of total leaf wax extracts…………………………………………..23
2.2.2. Preparation of epicuticular and intracuticular wax extracts…………………24

2.2.3. Qualitative and quantitative analyses of wax extracts………………………...24
2.2.4. Synthesis of reference compounds………………………………………………25
2.3. Results………………………………..…………………………………………………26
2.3.1. Structure elucidation of unknown compounds in A. arborescens leaf cuticular
wax……………………………………………………………………………………….26
2.3.2 Quantitative compositional analysis of total cuticular wax of A. arborescens
leaves.................................................................................................................................34
2.3.3. Distribution of A. arborescens leaf cuticular waxes between adaxial and
abaxial surfaces, as well as epicuticular and intracuticular layers…………..……...40
2.4. Discussion…………………..…………………………………………………………..44

2.4.1. Chain length distributions of ubiquitous wax constituents……………………44
2.4.2. Identification and chain length distributions of novel wax constituents……..47

2.4.3. Gradients between epi- and intracuticular wax layers………………………...52
Chapter 3: Composition of the cuticular waxes coating the adaxial side of Phyllostachys
aurea leaves: Identification of very-long-chain primary amides .............................................53
3.1. Introduction…………………..………………………………………………………...53
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3.2. Experimental……………………..…………………………………………………….57

3.2.1. Plant material and preparative thin layer chromatography (TLC)………….57

3.2.2. Preparation of epicuticular and intracuticular wax extracts…………………58

3.2.3. Derivatization reactions………………………………………………………….59
3.2.4. Gas chromatography (GC) analysis of wax extracts…………………………..59
3.2.5. Synthesis of C amide standard………………………………………………...61
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3.3. Results and discussion………………………………..………………………………..61
3.3.1. Identification of very-long-chain (VLC) fatty acid amides in the adaxial
cuticular waxes of P. aurea leaves………..………………...……………………….....62
3.3.2. Compound class gradients between the epicuticular and intracuticular layers
lining P. aurea leaves……..……………………………………………………...……..68
3.3.3. Individual compound gradients between the epicuticular and intracuticular
layers lining P. aurea leaves…………..………………………...……………………...72
3.3.4. Ester isomer profiles in epicuticular and intracuticular layers on P. aurea
leaves…………………………………………………………………………………….75
Chapter 4: Composition of cuticular waxes coating flag leaf blades and peduncles of
Triticum aestivum cv. Bethlehem………..…………………..…………………………………81

4.1. Introduction……………..……………………………………………………………...81
4.2. Experimental…………………………………………………………………………...85
4.2.1. Plant material and preparation of total wax extracts………………………….85
4.2.2. Derivatization reactions………………………………………………………….86
4.2.3. GC-MS and GC-FID analysis…………………………………………………...87
4.3. Results……………………………..……………………………………………………88
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4.3.1. Overall composition of wheat flag leaf blades and peduncles………………...88
4.3.2. Chain length distributions of common wheat compound classes……………..93
4.3.3. Chain length distributions of wheat wax esters………………………………..95

4.3.4. Chain length distributions of esterified alcohols and acids: ester isomer
compositions……………………………………………………………………….....…99
4.3.5. Composition of wheat wax polyketides and terpenoids……………………...107
4.4. Discussion……………..………………………………………………………………111
4.4.1. Wax composition differences between organs and new compound classes…111
4.4.2. Chain length distributions of ubiquitous wax compound classes……………113
4.4.3. Chain length and isomer distributions of aliphatic and aromatic esters……115
4.4.4. Chain length distributions of polyketide wax compound classes……………117
Chapter 5: Novel oxidized compounds and internally methyl-branched alkanes from
cuticular waxes of Triticum aestivum cv. Bethlehem…………………...……………………119
5.1. Introduction……………………………………..…………………………………….119
5.2. Experimental……………………………..…………………………………………...122
5.2.1. Plant material…………………………………………………………………...122
5.2.2. Chemicals………………………………………………………………………..122
5.2.3. Preparation of wax extracts……………………………………………………123
5.2.4. Preparative thin layer chromatography………………………………………123
5.2.5. Derivatization reactions………………………………………………………...124
5.2.6. Gas chromatography…………………………………………………………...125
5.3. Results…………………………………………..……………………………………..126
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5.3.1. TLC separation of cuticular waxes of T. aestivum flag leaf blades and
peduncles……………………………………………………………………………….126
5.3.2. Structure elucidation of compound classes A - C…………………………….128

5.3.3. Structure elucidation of compound classes D and E…………………………138

5.3.4. Structure elucidation of compound classes F and G…………………………144

5.3.5. Quantification of new compounds from cuticular waxes of T. aestivum cv.
Bethlehem flag leaf blades and peduncles…………………………………………...149

5.4. Discussion…………………………………………………………………………......153

5.4.1. Secondary alcohols, primary/secondary diols and diol esters in flag leaf
wax……………………………………………………………………………………...153
5.4.2. Hydroxy-2-alkanol esters and oxo-2-alkanol esters in flag leaf and peduncle
wax……………………………………………………………………………………...157
5.4.3. 4-Alkylbutan-4-olides and internally branched alkanes in flag leaf and
peduncle waxes………………………………………………………………………...159
Chapter 6: Novel polyketides and polyketide-derived compounds from cuticular waxes of
Triticum aestivum cv. Bethlehem …………………………………………….………………161

6.1. Introduction………………..………………………………………………………….161
6.2. Experimental………………..………………………………………………………...166
6.2.1. Plant material…………………………………………………………………...166
6.2.2. Wax extracts…………………………………………………………………….166
6.2.3. Preparative thin layer chromatography………………………………………167
6.2.4. Derivatization reactions………………………………………………………..167
6.2.5. Gas chromatography-mass spectrometry……………………………………..169
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